WGCNA of differentially expressed genes. A, Heatmap of relationships between genes with similar expression patterns in WGCNA modules and anthocyanin productions. Numbers in cells are the weight correlations (a measure of correlation of expression levels) between levels of specific anthocyanidins and individual modules, which consist of DEGs with similar expression patterns across samples and stages. P values of correlations are in parenthesis. Module names and the number of genes in modules are on the left of the heatmap. Anthocyanidins are indicated along the bottom of the heatmap. Cell color encodes signs of correlation (red with positive correlation; blue with negative correlation), while the intensity of the color indicates the magnitude of the correlation, as specified by the key to the right. B, Left graphs: Mean ± standard deviation (SD) of expression (as measured by FPKM) of two ABP-related genes (ACT, GSTF11) and one CBP-related gene (LCYE) at the four developmental stages (S1–S4) in the two species and the two hybrids (N = 48, 4-time points × 4 plant individuals × 3 biological replicates per individual). The r value correlations between expression level with pigment content. Different lowercase letters (a, ab, b, bc, c) above the bars indicate significant differences among taxa within a developmental stage as revealed by Duncan's multiple range test (P value < 0.017 for multiple comparisons). Means with the same letter (b and c) were not significantly different. Right graphs: the mean expression levels obtained from RT-qPCR for the same three genes in the two species (three replicates) at stage S1 (Student's t test, ***P value < 0.001). Y axis: Mean Expression (2^−ΔΔCt). Error bars represent the SD for three biological replicates in the two lotus species.